ABSTRACT
Numerous studies have demonstrated that FTO plays an important role in adipogenesis. Herein, we designed a small interfering RNA targeting FTO to knock down its endogenous expression and investigated its effects on the proliferation and differentiation of porcine intramuscular preadipocytes. Its possible mechanism was also investigated. We showed that FTO silencing significantly decreased the level of phospho-Histone H3 protein and inhibited the proliferation of porcine intramuscular preadipocytes. In addition, the expressions of peroxisome proliferators-activated receptor γ (PPARγ) and CAAT/enhancer binding protein (C/EBPα) were down-regulated, but the expression of ß-catenin was up-regulated, by FTO silencing. Of specific interest here was that LiCl, a Wnt/ß-catenin signaling specific activator, attenuated the FTO-induced upregulation of PPARγ and downregulation of ß-catenin. Collectively, our data demonstrated that FTO silence decreased the proliferation and differentiation of porcine intramuscular preadipocytes, and FTO affects the porcine intramuscular preadipocytes differentiation might be via Wnt/ß-catenin signaling pathway.
Subject(s)
Adipocytes/metabolism , Adipogenesis/physiology , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Wnt Signaling Pathway/physiology , Adipogenesis/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Animals , Cell Proliferation/genetics , Cell Proliferation/physiology , Cells, Cultured , Gene Knockdown Techniques , RNA, Small Interfering/genetics , Swine , Wnt Signaling Pathway/geneticsABSTRACT
Akirin2, a novel nuclear factor, plays an important role in myogenesis. To investigate the role of Akirin2 in proliferation and differentiation of porcine skeletal muscle satellite cells, Akirin2 overexpression and Akirin2 silence technologies were employed. Our results showed that overexpression of Akirin2 markedly enhanced the proliferation and differentiation of porcine skeletal muscle satellite cells, whereas silencing of Akirin2 got the opposite results. Furthermore, our results showed that Akirin2 affected proliferation and differentiation of porcine skeletal muscle satellite cells through extracellular-signal regulated kinase-1/2 (ERK1/2) and NFATc1 signaling pathways. These results indicate that Akirin2 can effectively promote skeletal muscle satellite cells proliferation and differentiation, acting through ERK1/2- and NFATc1-dependent mechanisms.
Subject(s)
Cell Differentiation/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NFATC Transcription Factors/metabolism , Repressor Proteins/genetics , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism , Signal Transduction , Animals , Cell Proliferation , Gene Expression , Gene Silencing , RNA, Messenger/genetics , Repressor Proteins/metabolism , SwineABSTRACT
The objective of this study was to evaluate the effect of Akirin2 on slow myosin heavy chain (slow MyHC, MyHC I) gene expression and its molecular mechanisms. In this study, we showed that the protein expression of Akirin2 in pig slow oxidative Psoas major muscle is higher than that in fast glycolytic tibialis anterior muscle, suggesting that Akirin2 may play a role in myofiber typing. Knockdown of Akirin2 decreased the MyHC I expression and the calcineurin (CaN) activity, and also decreased the expressions of NFATc1 and MCIP1.4. Conversely, overexpression of Akirin2 got the opposite results. Furthermore, inhibition of CaN or knockdown of NFATc1 attenuated Akirin2 overexpression-induced upregulation of MyHC I. Together, these results demonstrate that Akirin2 promotes MyHC I expression via CaN/NFATc1 signaling pathway in porcine skeletal muscle satellite cells.
Subject(s)
Gene Expression Regulation , Myosin Heavy Chains/genetics , NFATC Transcription Factors/metabolism , Nuclear Pore Complex Proteins/metabolism , Repressor Proteins/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Signal Transduction , Animals , Cells, Cultured , Female , Repressor Proteins/genetics , SwineABSTRACT
Dietary l-arginine supplementation decreases backfat thickness and increases intramuscular fat content in growing-finishing pigs, but the underlying mechanisms are unknown. In this study, the effect of arginine on differentiation of porcine intramuscular preadipocytes was investigated in vitro. We showed that the mRNA and protein expressions of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α were upregulated by arginine supplementation. Furthermore, the intracellular triglyceride level was increased by arginine supplementation. We also showed that activation of the Wnt/ß-catenin signal pathway by using lithium chloride (LiCl) significantly attenuated arginine-induced upregulation of PPARγ and increased the phospho-ß-catenin level. These findings suggested that arginine promotes porcine intramuscular preadipocyte differentiation, which might be via repressing the Wnt/ß-catenin signaling pathway.
Subject(s)
Adipocytes/metabolism , Adipogenesis , Arginine/metabolism , CCAAT-Enhancer-Binding Protein-alpha/metabolism , PPAR gamma/metabolism , Wnt Signaling Pathway , Adipocytes/cytology , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , Cells, Cultured , Female , Male , PPAR gamma/genetics , Swine , Triglycerides/metabolismABSTRACT
Fatty acid transport protein 1 (FATP1) plays an important role in the fatty acid transmembrane transport and fat deposition. However, its role in porcine intramuscular preadipocytes proliferation and differentiation remain poorly understood. Here, we examined the effects of pFATP1 on porcine intramuscular preadipocytes proliferation and differentiation. Overexpression of pFATP1 in porcine intramuscular preadipocytes significantly promoted the proliferation of porcine intramuscular preadipocytes, and also significantly upregulated the expressions of peroxisome proliferator-activated receptor γ, CCAAT enhancer binding protein α, lipoprotein lipase, fatty acid synthetase and perilipin 1. Moreover, overexpression of pFATP1 in porcine intramuscular preadipocytes significantly increased fat accumulation and downregulated ß-catenin protein expression. Overall, our results indicated that pFATP1 played an important role in porcine intramuscular preadipocytes proliferation and differentiation, and it might promote adipogenesis in porcine intramuscular preadipocytes by repressing Wnt/ß-catenin signaling pathway.
Subject(s)
Adipocytes/cytology , Adipogenesis/genetics , Cell Differentiation/genetics , Cell Proliferation/genetics , Fatty Acid Transport Proteins/physiology , Muscles/cytology , Animals , Cell Membrane/metabolism , Cells, Cultured , Fatty Acid Transport Proteins/genetics , Fatty Acids/metabolism , Gene Expression , Male , Swine , Wnt Signaling Pathway/geneticsABSTRACT
Phosphotyrosine interaction domain containing 1 (PID1), a recently identified gene involved in obesity-associated insulin resistance, plays an important role in fat deposition. However, its effect on porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, the plasmid pcDNA3.1(+)-pPID1 was transfected into porcine intramuscular preadipocytes with Lipofectamine 3000 reagent to over-express porcine PID1 (pPID1). Over-expression of pPID1 significantly promoted porcine intramuscular preadipocyte proliferation. Expression of pPID1 mRNA was significantly increased upon porcine intramuscular preadipocyte differentiation. Indirect fluorescent immunocytochemistry demonstrated that pPID1 protein was localized predominantly in the nucleus of porcine intramuscular preadipocyte. The mRNA levels of peroxisome proliferators-activated receptor γ, CCAAT/enhancer binding protein α and lipoprotein lipase were significantly increased by pPID1 over-expression. Over-expression of pPID1 also led to an increase in lipid accumulation which was detected by Oil Red O staining, and significantly increased the intramuscular triacylglycerol content. These results indicate that pPID1 may play a role in enhancing porcine intramuscular preadipocyte proliferation and differentiation.
Subject(s)
Adipocytes/cytology , Carrier Proteins/genetics , Cell Differentiation/genetics , Cell Proliferation/genetics , Animals , Cells, Cultured , Male , Muscle, Skeletal/cytology , SwineABSTRACT
The fat mass and obesity associated (FTO) gene plays an important role in adipogenesis. However, its function during porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, we prepared the antiserum against porcine FTO (pFTO), which was used to determine its subcellular localization and tissue distribution. Our data indicated that pFTO was localized predominantly in the nucleus. Real-time quantitative PCR and western blot analysis showed that pFTO was highly expressed in the lung and subcutaneous adipose tissue. Overexpression of pFTO in porcine intramuscular preadipocytes significantly promoted cell proliferation and lipid deposition. Furthermore, overexpression of pFTO in differentiating porcine intramuscular preadipocytes also significantly increased the mRNA levels of adipocyte differentiation transcription factors peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and fatty acid synthase (FAS). Our findings provide the first functional evidence to reveal a role of pFTO in porcine intramuscular preadipocyte proliferation and differentiation.
Subject(s)
Body Fat Distribution , Cell Differentiation/genetics , Cell Proliferation/genetics , Proteins/analysis , Subcutaneous Fat , Swine/genetics , Adipocytes , Animals , Cells, Cultured , Female , Proteins/metabolism , Proteins/physiology , Swine/anatomy & histologyABSTRACT
Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZaA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZaFA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0-6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2 +, Mg2 +, Cu2 +, Zn2 +, Mn2 +, Na+ and EDTA, but activated by Fe2 +. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions The two-copy strain GSKZaFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris.
Subject(s)
Pichia/enzymology , Aspergillus niger/enzymology , Carboxylic Ester Hydrolases/metabolism , Yeasts , Blotting, WesternABSTRACT
Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway.
